CN117630242A - Method for separating and detecting impurity a in clotrimazole intermediate by high performance liquid chromatography - Google Patents
Method for separating and detecting impurity a in clotrimazole intermediate by high performance liquid chromatography Download PDFInfo
- Publication number
- CN117630242A CN117630242A CN202311746922.2A CN202311746922A CN117630242A CN 117630242 A CN117630242 A CN 117630242A CN 202311746922 A CN202311746922 A CN 202311746922A CN 117630242 A CN117630242 A CN 117630242A
- Authority
- CN
- China
- Prior art keywords
- impurity
- mobile phase
- clotrimazole
- solution
- volume ratio
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000012535 impurity Substances 0.000 title claims abstract description 72
- 238000000034 method Methods 0.000 title claims abstract description 43
- VNFPBHJOKIVQEB-UHFFFAOYSA-N clotrimazole Chemical compound ClC1=CC=CC=C1C(N1C=NC=C1)(C=1C=CC=CC=1)C1=CC=CC=C1 VNFPBHJOKIVQEB-UHFFFAOYSA-N 0.000 title claims abstract description 38
- 229960004022 clotrimazole Drugs 0.000 title claims abstract description 38
- 238000004128 high performance liquid chromatography Methods 0.000 title abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 28
- 239000000523 sample Substances 0.000 claims abstract description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 12
- KTVAHLGKTSPDOG-UHFFFAOYSA-N TRAM-3 Chemical compound C=1C=CC=CC=1C(C=1C(=CC=CC=1)Cl)(O)C1=CC=CC=C1 KTVAHLGKTSPDOG-UHFFFAOYSA-N 0.000 claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- JFLSOKIMYBSASW-UHFFFAOYSA-N 1-chloro-2-[chloro(diphenyl)methyl]benzene Chemical compound ClC1=CC=CC=C1C(Cl)(C=1C=CC=CC=1)C1=CC=CC=C1 JFLSOKIMYBSASW-UHFFFAOYSA-N 0.000 claims abstract description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000012488 sample solution Substances 0.000 claims abstract description 10
- 238000010828 elution Methods 0.000 claims abstract description 9
- 238000010812 external standard method Methods 0.000 claims abstract description 6
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 5
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 35
- 239000002904 solvent Substances 0.000 claims description 28
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 21
- 238000001212 derivatisation Methods 0.000 claims description 15
- 239000011259 mixed solution Substances 0.000 claims description 15
- 239000013558 reference substance Substances 0.000 claims description 11
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 claims description 8
- 230000014759 maintenance of location Effects 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 230000006399 behavior Effects 0.000 claims description 2
- 238000003908 quality control method Methods 0.000 abstract description 3
- 238000004451 qualitative analysis Methods 0.000 abstract 1
- 238000004445 quantitative analysis Methods 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 43
- 238000007865 diluting Methods 0.000 description 23
- 239000011550 stock solution Substances 0.000 description 18
- 239000000047 product Substances 0.000 description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Natural products CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- 238000005303 weighing Methods 0.000 description 7
- 239000012467 final product Substances 0.000 description 6
- 238000001816 cooling Methods 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- KAXCEFLQAYFJKV-UHFFFAOYSA-N 2-anilinoacetonitrile Chemical compound N#CCNC1=CC=CC=C1 KAXCEFLQAYFJKV-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 239000012490 blank solution Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- FYSNRJHAOHDILO-UHFFFAOYSA-N thionyl chloride Chemical compound ClS(Cl)=O FYSNRJHAOHDILO-UHFFFAOYSA-N 0.000 description 2
- VMHYWKBKHMYRNF-UHFFFAOYSA-N (2-chlorophenyl)-phenylmethanone Chemical compound ClC1=CC=CC=C1C(=O)C1=CC=CC=C1 VMHYWKBKHMYRNF-UHFFFAOYSA-N 0.000 description 1
- SYRDONZIYFYBHS-UHFFFAOYSA-N (3-chlorophenyl)-diphenylmethanol Chemical compound C=1C=CC=CC=1C(C=1C=C(Cl)C=CC=1)(O)C1=CC=CC=C1 SYRDONZIYFYBHS-UHFFFAOYSA-N 0.000 description 1
- KHVJXWFCBMQXLH-UHFFFAOYSA-N (4-chlorophenyl)-diphenylmethanol Chemical compound C=1C=CC=CC=1C(C=1C=CC(Cl)=CC=1)(O)C1=CC=CC=C1 KHVJXWFCBMQXLH-UHFFFAOYSA-N 0.000 description 1
- IKKSPFNZXBWDQA-UHFFFAOYSA-N 1-benzyl-2-chlorobenzene Chemical compound ClC1=CC=CC=C1CC1=CC=CC=C1 IKKSPFNZXBWDQA-UHFFFAOYSA-N 0.000 description 1
- DGRVQOKCSKDWIH-UHFFFAOYSA-N 1-chloro-2-(trifluoromethyl)benzene Chemical compound FC(F)(F)C1=CC=CC=C1Cl DGRVQOKCSKDWIH-UHFFFAOYSA-N 0.000 description 1
- PHRLCYSMTGTXMM-UHFFFAOYSA-N 1-chloro-2-[dichloro(phenyl)methyl]benzene Chemical compound ClC1=CC=CC=C1C(Cl)(Cl)C1=CC=CC=C1 PHRLCYSMTGTXMM-UHFFFAOYSA-N 0.000 description 1
- IBSQPLPBRSHTTG-UHFFFAOYSA-N 1-chloro-2-methylbenzene Chemical compound CC1=CC=CC=C1Cl IBSQPLPBRSHTTG-UHFFFAOYSA-N 0.000 description 1
- JIOMOSMOFRFEGZ-UHFFFAOYSA-N 1-chloro-3-[chloro(diphenyl)methyl]benzene Chemical compound ClC1=CC=CC(C(Cl)(C=2C=CC=CC=2)C=2C=CC=CC=2)=C1 JIOMOSMOFRFEGZ-UHFFFAOYSA-N 0.000 description 1
- LMSWYOHFMJDYAF-UHFFFAOYSA-N 1-chloro-4-[chloro(diphenyl)methyl]benzene Chemical compound C1=CC(Cl)=CC=C1C(Cl)(C=1C=CC=CC=1)C1=CC=CC=C1 LMSWYOHFMJDYAF-UHFFFAOYSA-N 0.000 description 1
- HZJKXKUJVSEEFU-UHFFFAOYSA-N 2-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)hexanenitrile Chemical compound C=1C=C(Cl)C=CC=1C(CCCC)(C#N)CN1C=NC=N1 HZJKXKUJVSEEFU-UHFFFAOYSA-N 0.000 description 1
- IKCLCGXPQILATA-UHFFFAOYSA-N 2-chlorobenzoic acid Chemical compound OC(=O)C1=CC=CC=C1Cl IKCLCGXPQILATA-UHFFFAOYSA-N 0.000 description 1
- ONIKNECPXCLUHT-UHFFFAOYSA-N 2-chlorobenzoyl chloride Chemical compound ClC(=O)C1=CC=CC=C1Cl ONIKNECPXCLUHT-UHFFFAOYSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- KZBUYRJDOAKODT-UHFFFAOYSA-N Chlorine Chemical compound ClCl KZBUYRJDOAKODT-UHFFFAOYSA-N 0.000 description 1
- 241001337994 Cryptococcus <scale insect> Species 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 238000005727 Friedel-Crafts reaction Methods 0.000 description 1
- 239000005811 Myclobutanil Substances 0.000 description 1
- 241000893976 Nannizzia gypsea Species 0.000 description 1
- 241000223229 Trichophyton rubrum Species 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical group O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 description 1
- 150000001448 anilines Chemical class 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940095731 candida albicans Drugs 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- LZTRCELOJRDYMQ-UHFFFAOYSA-N triphenylmethanol Chemical compound C=1C=CC=CC=1C(C=1C=CC=CC=1)(O)C1=CC=CC=C1 LZTRCELOJRDYMQ-UHFFFAOYSA-N 0.000 description 1
- JBWKIWSBJXDJDT-UHFFFAOYSA-N triphenylmethyl chloride Chemical compound C=1C=CC=CC=1C(C=1C=CC=CC=1)(Cl)C1=CC=CC=C1 JBWKIWSBJXDJDT-UHFFFAOYSA-N 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the technical field of pharmaceutical analytical chemistry, and particularly relates to a method for separating and detecting impurity a in a clotrimazole intermediate by using a high performance liquid chromatography. The clotrimazole intermediate is specifically o-chlorophenyl-diphenyl-chloromethane, and the impurity a is diphenyl- (2-chlorophenyl) methanol. The high performance liquid chromatography comprises: treating a sample to be detected by adopting a derivative reagent to obtain a sample solution; octadecylsilane chemically bonded silica is used as a chromatographic column stationary phase, water is used as a mobile phase A, methanol is used as a mobile phase B, and impurities a and derivatives of a clotrimazole intermediate are separated through linear gradient elution; then entering a detector for detection; and (3) carrying out qualitative and quantitative analysis on the impurity a by adopting an external standard method according to the chromatographic result. The method has the advantages of strong specificity, good durability and low detection limit, and has important significance for quality control of the o-chlorophenyl-diphenyl-chloromethane and clotrimazole products.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical analytical chemistry, and particularly relates to a method for separating and detecting impurity a in a clotrimazole intermediate by using a high performance liquid chromatography.
Background
Clotrimazole is a high-efficiency and low-cost broad-spectrum antifungal drug and has remarkable inhibition effect on candida albicans, trichophyton rubrum, trichophyton gypseum, novel cryptococcus, aspergillus, algae bacteria and the like. O-chlorophenyl-diphenyl-chloromethane is a key intermediate for synthesizing clotrimazole, and the synthetic route is mainly prepared by taking O-chlorotrifluorotoluene as a raw material, performing Friedel-crafts reaction in benzene under the catalysis of anhydrous aluminum trichloride, and performing chlorination reaction with thionyl chloride; and secondly, diphenyl- (2-chlorophenyl) methanol and dimethyl sulfoxide are subjected to reflux reaction for 5 hours at 90 ℃ under the condition of introducing chlorine gas. The o-chlorophenyl-diphenyl-chloromethane is extremely easily hydrolyzed, and diphenyl- (2-chlorophenyl) methanol, i.e., pharmacopoeia impurity i, is formed if the preservation conditions are improper, and the impurity is described in USP, EP, JP, BP. Pharmacopoeia prescribes that this impurity limit must be controlled below 0.3% in clotrimazole and that the relevant formulation must be controlled below 1.0% and it can be seen that the control of the impurity diphenyl- (2-chlorophenyl) methanol in this product is very important, which is related to whether the final product clotrimazole is acceptable.
O-chlorophenyl-diphenyl-chloromethane, english chemical name 1-chlor-2- (chlorodiphenylmethyl) benzene, molecular formula C 19 H 14 Cl 2 The molecular weight is 313.22, CAS number is 42074-68-0, and the structural formula is shown in formula I. The structural formula of diphenyl- (2-chlorophenyl) methanol (impurity a) is shown as formula II.
At present, no analysis method for diphenyl- (2-chlorophenyl) methanol in o-chlorophenyl-diphenyl-chloromethane is searched, and therefore, a new method for detecting and controlling the diphenyl- (2-chlorophenyl) methanol in the o-chlorophenyl-diphenyl-chloromethane is needed.
Disclosure of Invention
Therefore, one of the purposes of the invention is to provide a method for separating impurity a in a clotrimazole intermediate by utilizing a pre-column derivatization method, which can realize the separation of impurity a in the clotrimazole intermediate within 30 minutes or more than 30 minutes and has the characteristics of strong specificity, good durability and short analysis time.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the method for separating impurity a in the clotrimazole intermediate by utilizing a pre-column derivatization method, wherein the clotrimazole intermediate is o-chlorophenyl-diphenyl-chloromethane, and the structural formula of the clotrimazole intermediate is shown as formula I; the impurity a is diphenyl- (2-chlorophenyl) methanol, and the structural formula of the impurity a is shown as a formula II; the pre-column derivatization method comprises: treating a sample to be detected by adopting a derivative reagent to obtain a sample solution; octadecylsilane chemically bonded silica is used as a chromatographic column stationary phase, a mobile phase A and a mobile phase B are used as mobile phases, the mobile phase A is water, and the mobile phase B is methanol; separating the impurity a and the derivative of the clotrimazole intermediate by gradient elution;
according to the research of the invention, if the liquid phase system is directly fed, the impurity a and the o-chlorophenyl-diphenyl-chloromethane of the product can show a peak at the same position, and the pharmacopoeia related method is only suitable for detecting the impurity in clotrimazole and is not suitable for the product. Therefore, the aniline derivative method is adopted to carry out pre-column derivatization on the product, so that the product (derivative) and diphenyl- (2-chlorophenyl) methanol are effectively separated. The method has important significance for synthesizing and controlling the quality of the clotrimazole.
Further, the derivatizing agent is a mixed solution of aniline and acetonitrile, wherein the volume ratio of aniline to acetonitrile is 1:1.
further, the temperature of the derivatization treatment is 40-60 ℃, preferably 50 ℃.
Further, the gradient elution procedure was as follows:
setting the volume ratio of the mobile phase A to the mobile phase B to be 25-40 in 0 min: 60-75;
setting the volume ratio of the mobile phase A to the mobile phase B to be 25-40 in 2 minutes: 60-75;
setting the volume ratio of the mobile phase A to the mobile phase B to be 10-20 in 20 minutes: 80-90;
setting the volume ratio of the mobile phase A to the mobile phase B to be 25-40 in 21 minutes: 60-75;
setting the volume ratio of the mobile phase A to the mobile phase B to be 25-40 in 30 minutes: 60-75.
Preferably, the gradient elution procedure is as follows:
setting the volume ratio of the mobile phase A to the mobile phase B to be 28-32 in 0 min: 68-72;
setting the volume ratio of the mobile phase A to the mobile phase B to be 28-32 in 2 minutes: 68-72;
at 20 minutes, the volume ratio of mobile phase a to mobile phase B was set to 10:90;
setting the volume ratio of the mobile phase A to the mobile phase B to be 28-32 in 21 minutes: 68-72;
setting the volume ratio of the mobile phase A to the mobile phase B to be 28-32 in 30 minutes: 68-72.
Most preferably, the gradient elution procedure is as follows:
at 0 minutes, the volume ratio of mobile phase a and mobile phase B was set to 30:70;
at 2 minutes, the volume ratio of mobile phase a and mobile phase B was set to 30:70;
at 20 minutes, the volume ratio of mobile phase a to mobile phase B was set to 10:90;
at 21 minutes, the volume ratio of mobile phase a and mobile phase B was set to 30:70;
at 30 minutes, the volume ratio of mobile phase a to mobile phase B was set to 30:70.
further, the column flow rate is 0.8 to 1.5ml/min, preferably 0.9 to 1.1ml/min, more preferably 1.0ml/min; the column temperature is 15-25 ℃, preferably 18-22 ℃, more preferably 20 ℃.
Preferably, the chromatographic column employs GL Sciences AX-C18; the column size was 4.6mm X250 mm,5 μm.
Preferably, the sample volume is 10. Mu.l.
The second purpose of the invention is to provide a method for detecting whether the clotrimazole intermediate contains the impurity a, the method successfully realizes the qualitative detection of the impurity a in the clotrimazole intermediate, and the detection limit is as low as 0.0904 mug/ml, and the method has higher sensitivity.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for detecting whether the clotrimazole intermediate contains an impurity a, and separating the impurity a from the clotrimazole intermediate by using the method; and the sample enters a detector with the detection wavelength of 225+/-5 nm for detection to obtain a chromatogram; and judging whether the clotrimazole intermediate contains the impurity a according to the consistency of the chromatographic behaviors of the detection product and the reference product.
Preferably, the detection wavelength is 225nm.
By the retention time, the impurity a can be characterized.
Further, the retention time was 11.9.+ -. 0.5min, and it was judged that the impurity a; the retention time was 19.8.+ -. 0.5min, which was judged as a derivative of the clotrimazole intermediate.
The invention further aims to provide a method for measuring the content of the impurity a in the clotrimazole intermediate, which is simple to operate and good in reproducibility, and the quantitative detection of the impurity a in the clotrimazole intermediate is successfully realized.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the method for determining the content of impurity a in the clotrimazole intermediate comprises the following steps:
(1) Treating a sample to be detected by adopting a derivative reagent, and preparing a sample solution and a reference substance solution by adopting a solvent;
(2) Detecting the sample solution and the reference solution by the method of any one of claims 6 to 8, and obtaining a chromatogram;
(3) And (3) obtaining a chromatogram according to the step (2), and calculating the content of the impurity a by adopting an external standard method according to the peak area.
Further, the solvent is acetonitrile water solution, wherein the volume ratio of acetonitrile to water is 95:5.
Further, in the concentration range of 0.3-30.1 μg/ml, the linear equation of impurity a is y=0.424 x+0.0053, r=1.0000; where X is the X-axis, representing concentration, and Y is the Y-axis, representing peak area.
As a preferred implementation method:
1. taking the product, such as 75mg, precisely weighing, placing into a 10ml measuring flask, immediately adding 5ml of derivatization reagent, carrying out ultrasonic treatment to dissolve, placing into a 50 ℃ water bath to heat for 10 minutes, cooling, diluting to a scale with a solvent, shaking uniformly, precisely transferring a proper amount, and diluting with the solvent to prepare a solution with about 0.3mg in each 1ml as a sample solution;
2. precisely weighing a proper amount of impurity a reference substance, dissolving in a solvent, quantitatively diluting to obtain a solution containing about 9 mug per 1ml, and taking the solution as a reference substance solution;
3. precisely measuring the solution of the sample and the solution of the reference substance, respectively injecting into a liquid chromatograph, and recording chromatograms; and the impurity content was calculated as peak area according to the external standard method.
Further, preparing a standard substance and detecting to obtain a chromatogram of the standard substance; and (5) testing the chromatograms obtained by the samples, and calculating the content according to an external standard method by using the peak area.
The standard substance is the known component content, and the component content of the test sample is unknown.
The obtained standard sample chromatograms can be stored in a database and used as quality control map materials in an intelligent pharmaceutical factory, and the quality control map materials are used for helping a robot to judge whether products produced by the robot are qualified or not, and quantitatively improved analysis suggestions are provided for unqualified products.
Preferably, if the impurity a content is less than 3.0%, the sample is qualified.
The invention has the beneficial effects that:
1. according to the invention, the aniline-acetonitrile mixed solution is selected as the derivative liquid for heating and derivatization, the chromatographic column using octadecylsilane chemically bonded silica as the filler and the water-methanol elution system realize the detection of diphenyl- (2-chlorophenyl) methanol within 30 minutes, and the detection time is short.
2. The detection method provided by the invention can meet the requirements on the separation degree with other impurities possibly existing in the product, is not interfered by other impurities and test products, can accurately and efficiently quantify the impurity a, and has stronger specificity and durability; and simultaneously, other detected impurities in the mixed solution can be quantified according to a peak area normalization method.
3. The method for detecting the impurity a in the myclobutanil intermediate has the characteristics of low detection limit of 0.0904 mug/ml and high sensitivity.
Drawings
FIG. 1 is an HPLC diagram of a mixed solution;
FIG. 2 is an HPLC plot of durability versus flow rate of 0.9 ml/min-mixed solution;
FIG. 3 is an HPLC plot of durability versus flow rate of 1.1 ml/min-mixed solution;
FIG. 4 is an HPLC plot of durability versus column temperature of 18℃for the mixed solution;
FIG. 5 is an HPLC plot of durability versus column temperature 22℃for the mixed solution;
FIG. 6 is an HPLC plot of durability-initial ratio of mobile phase (68% organic phase) -mixed solution;
FIG. 7 is an HPLC plot of durability-initial ratio of mobile phase (72% organic phase) -mixed solution;
FIG. 8 is an HPLC chart of the detection limit solution;
FIG. 9 is an HPLC plot of linear solution # 1;
FIG. 10 is an HPLC plot of linear solution # 2;
FIG. 11 is an HPLC plot of linear solution 3#;
FIG. 12 is an HPLC plot of linear solution # 4;
FIG. 13 is an HPLC plot of linear solution # 5;
FIG. 14 is an HPLC plot of linear solution 6;
FIG. 15 is an HPLC plot of linear solution # 7;
fig. 16 is a linear relationship diagram.
Detailed Description
The technical scheme of the present invention will be further clearly and completely described in connection with specific embodiments. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. Therefore, all other embodiments obtained by those skilled in the art without undue burden are within the scope of the invention based on the embodiments of the present invention.
In the embodiment of the invention, the high performance liquid chromatography for separating and detecting the impurity a in the clotrimazole intermediate comprises the following steps:
1. chromatographic conditions
As shown in tables 1 and 2.
TABLE 1 chromatographic conditions
TABLE 2 gradient elution program table
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 25～40 | 60～75 |
| 2 | 25～40 | 60～75 |
| 20 | 10～20 | 80～90 |
| 21 | 25～40 | 60～75 |
| 30 | 25～40 | 60～75 |
2. Solution preparation
Test solution: taking 75mg of the product, precisely weighing, placing into a 10ml measuring flask, immediately adding 5ml of derivatization reagent, carrying out ultrasonic treatment to dissolve, placing into a water bath at 50 ℃ for heating for 10 minutes, cooling, diluting to a scale with a solvent, shaking uniformly, precisely transferring a proper amount, and diluting with the solvent to prepare a solution containing about 0.3mg of the derivatization reagent per 1 ml.
The reference substance solution is precisely weighed a proper amount of the reference substance of impurity a, dissolved by a solvent and quantitatively diluted to prepare a solution with the concentration of about 9 mug per 1 ml.
3. Test method
Precisely measuring the sample solution and the reference substance solution, respectively injecting into a liquid chromatograph, and recording the chromatograms. The impurity a is less than 3.0 percent calculated by the external standard method according to the peak area.
In the embodiment of the invention, Z1c is impurity a in the map result measured in the embodiment.
In the embodiment of the invention, the detection is carried out according to the following chromatographic conditions unless otherwise indicated: octadecylsilane chemically bonded silica is used as filler (GL Sciences AX-C18, 4.6mm.times.250 mm,5 μm, or column with comparable performance); linear gradient elution was performed according to table 3 with water as mobile phase a and methanol as mobile phase B; the flow rate is 1.0ml per minute; the column temperature is 20 ℃; the detection wavelength is 225nm; the sample volume was 10. Mu.l.
TABLE 3 gradient elution program table
EXAMPLE 1 specificity
1. Preparation of test reagents
Derivatizing agent (used on the day): aniline-acetonitrile (1:1).
Blank solution: transferring derivatization reagent 5ml, placing into a 10ml measuring flask, heating in water bath at 50deg.C for 10 min, cooling, diluting with solvent to scale, shaking, precisely transferring 1ml, placing into a 25ml measuring flask, diluting with solvent to scale, and shaking.
Sample stock solution: precisely weighing about 75mg of the product, placing into a 10ml measuring flask, immediately adding 5ml of derivatization reagent, dissolving by ultrasound, heating in water bath at 50deg.C for 10 min, cooling, diluting to scale with solvent, and shaking.
Test solution: precisely transferring 1ml of sample stock solution, placing into a 25ml measuring flask, diluting with solvent to scale, and shaking.
Impurity a stock solution: accurately weighing 30mg of impurity a reference substance, placing into a 50ml measuring flask, adding ethanol solvent for dissolving and diluting to scale, and shaking; and precisely removing 2.5ml of impurity a stock solution, placing into a 50ml measuring flask, diluting to scale with solvent, and shaking to obtain the final product. (10%)
Impurity a positioning solution: and precisely removing 3ml of impurity a stock solution, placing in a 10ml measuring flask, diluting to scale with solvent, and shaking to obtain the final product.
Other mixed stock solutions: removing impurities of o-chlorotoluene, 2-chlorobenzoic acid, triphenylchloromethane, triphenylmethanol, 1-chloro-3- (chlorodiphenylmethyl) benzene, diphenyl- (3-chlorophenyl) methanol, 1-benzyl-2-chlorobenzene, 1' - [ (2-chlorophenyl) methylene ] diphenyl, 2-chlorobenzoyl chloride (5 mg) respectively, and impurities of 1-chloro-2- [ dichloro (phenyl) methyl ] benzene, 1-chloro-4- (chlorodiphenylmethyl) benzene, diphenyl- (4-chlorophenyl) methanol and 2-chlorobenzophenone (10 mg) respectively, placing into 50ml measuring flask, immediately adding 25ml of derivatization reagent, ultrasonically dissolving, placing into 50 ℃ water bath, heating for 10 minutes, cooling, diluting to scale with solvent, shaking, precisely removing 1ml, placing into 10ml measuring flask, diluting to scale with solvent, shaking to obtain the product.
Other impurity mixed solution: precisely transferring 1.5ml of mixed stock solution of other impurities, placing into a 50ml measuring flask, diluting to scale with solvent, and shaking.
Mixing solution: precisely transferring 0.4ml of sample stock solution, placing into a 10ml measuring flask, adding 3ml of reference stock solution and 0.3ml of other impurity mixed stock solution, diluting with solvent to scale, and shaking.
2. Detection of
Taking 10 mu l of each of the blank solution, the sample solution, the impurity a positioning solution, the other impurity mixed solution and the mixed solution, respectively injecting into a liquid chromatograph, and recording a chromatogram. The test results are shown in FIG. 1 and Table 4 below, in FIG. 1, the fourth peak is impurity a, and the highest peak is the derivative of the main component.
TABLE 4 specificity test results Table
Example 2 durability
The "example 1. Proprietary" mixed solution was used, and each chromatographic condition (including column flow.+ -. 0.1 ml/min, column temperature.+ -. 2 ℃ C., mobile phase B initial ratio.+ -. 2%) was adjusted finely to examine the degree of separation between peaks.
The test results are shown in fig. 2-7 and table 5 below, and the peak-to-peak separation of each component is greater than 1.5 when the flow rate, column temperature and mobile phase ratio slightly fluctuate, indicating that the invention has good durability.
TABLE 5 chromatographic System durability determination results
Example 3 detection Limit
1. Solution preparation
Detection limit stock solution: accurately weighing 30mg of impurity a reference substance, placing in a 50ml measuring flask, adding ethanol solvent for dissolving and diluting to scale, and shaking; and precisely removing 2.5ml of impurity a stock solution, placing into a 50ml measuring flask, diluting to scale with solvent, and shaking to obtain the final product.
Detection limit solution: and precisely transferring 0.3ml of the detection limit stock solution, placing into a 100ml measuring flask, diluting to a scale with a solvent, and shaking uniformly to obtain the final product.
2. Detection of
And precisely measuring 10 mu l of detection limit solution, continuously injecting the sample for 3 times, recording a chromatogram, and calculating the signal to noise ratio of each peak. The results are shown in FIG. 8 and Table 6 below.
TABLE 6 detection limit test results
EXAMPLE 4 Linear
1. Preparing a solution to be tested
Linear stock solution: accurately weighing 30mg of impurity a reference substance, placing into a 50ml measuring flask, adding ethanol solvent for dissolving and diluting to scale, and shaking; and precisely removing 2.5ml of impurity a stock solution, placing into a 50ml measuring flask, diluting to scale with solvent, and shaking to obtain the final product.
Linear solutions 1# to 3#: precisely transferring 1ml of each linear stock solution, respectively placing into 100ml, 25ml and 10ml measuring flask, diluting to scale with solvent, and shaking.
Linear solutions 4-6 #: precisely transferring linear stock solution 3ml, 5ml and 8ml, placing into 3 different 10ml measuring bottles, diluting to scale with solvent, and shaking.
Linear solution # 7: i.e., a linear stock solution.
2. Detection of
Taking the linear solutions, sequentially sampling from low to high, recording a chromatogram, and calculating a regression equation and a correlation coefficient by using a least square method to linearly regress the peak area Y by the concentration X (mug/ml). The test results are shown in FIGS. 9-16 and Table 7 below.
TABLE 7 Linear test results Table
Claims (10)
1. The method for separating impurity a in the clotrimazole intermediate by utilizing a pre-column derivatization method is characterized in that the clotrimazole intermediate is o-chlorophenyl-diphenyl-chloromethane, and the structural formula of the clotrimazole intermediate is shown as formula I; the impurity a is diphenyl- (2-chlorophenyl) methanol, and the structural formula of the impurity a is shown as a formula II; the pre-column derivatization method comprises: treating a sample to be detected by adopting a derivative reagent to obtain a sample solution; octadecylsilane chemically bonded silica is used as a chromatographic column stationary phase, a mobile phase A and a mobile phase B are used as mobile phases, the mobile phase A is water, and the mobile phase B is methanol; separating the impurity a and the derivative of the clotrimazole intermediate by gradient elution;
2. the method according to claim 1, wherein the derivatizing agent is a mixed solution of aniline and acetonitrile, wherein the volume ratio of aniline to acetonitrile is 1:1.
3. the method according to claim 1, wherein the temperature of the derivatization treatment is 40-60 ℃.
4. The method of claim 1, wherein the gradient elution procedure is as follows:
setting the volume ratio of the mobile phase A to the mobile phase B to be 25-40 in 0 min: 60-75;
setting the volume ratio of the mobile phase A to the mobile phase B to be 25-40 in 2 minutes: 60-75;
setting the volume ratio of the mobile phase A to the mobile phase B to be 10-20 in 20 minutes: 80-90;
setting the volume ratio of the mobile phase A to the mobile phase B to be 25-40 in 21 minutes: 60-75;
setting the volume ratio of the mobile phase A to the mobile phase B to be 25-40 in 30 minutes: 60-75.
5. The method according to claim 1, wherein the column flow rate is 0.8-1.5ml/min; the column temperature is 15-25 ℃.
6. A method for detecting the presence of impurity a in a clotrimazole intermediate, characterized in that the impurity a is separated from the clotrimazole intermediate by the method according to any one of claims 1 to 5; and the sample enters a detector with the detection wavelength of 225+/-5 nm for detection to obtain a chromatogram; and judging whether the clotrimazole intermediate contains the impurity a according to the consistency of the chromatographic behaviors of the detection product and the reference product.
7. The method according to claim 6, wherein the retention time is 11.9±0.5min, and the impurity a is determined; the retention time was 19.8.+ -. 0.5min, which was judged as a derivative of the clotrimazole intermediate.
8. The method for determining the content of impurity a in the clotrimazole intermediate is characterized by comprising the following steps:
(1) Treating a sample to be detected by adopting a derivative reagent, and preparing a sample solution and a reference substance solution by adopting a solvent;
(2) Detecting the sample solution and the control solution by the method of any one of claims 6 to 7, and obtaining a chromatogram;
(3) And (3) obtaining a chromatogram according to the step (2), and calculating the content of the impurity a by adopting an external standard method according to the peak area.
9. The method of claim 8, wherein the solvent is an aqueous acetonitrile solution, wherein the volume ratio of acetonitrile to water is 95:5.
10. The method of claim 8, wherein the linear equation for impurity a is y = 0.424x+0.0053, r = 1.0000, in the concentration range of 0.3-30.1 μg/ml; where X is the X-axis, representing concentration, and Y is the Y-axis, representing peak area.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311746922.2A CN117630242A (en) | 2023-12-18 | 2023-12-18 | Method for separating and detecting impurity a in clotrimazole intermediate by high performance liquid chromatography |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311746922.2A CN117630242A (en) | 2023-12-18 | 2023-12-18 | Method for separating and detecting impurity a in clotrimazole intermediate by high performance liquid chromatography |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117630242A true CN117630242A (en) | 2024-03-01 |
Family
ID=90032149
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311746922.2A Pending CN117630242A (en) | 2023-12-18 | 2023-12-18 | Method for separating and detecting impurity a in clotrimazole intermediate by high performance liquid chromatography |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117630242A (en) |
-
2023
- 2023-12-18 CN CN202311746922.2A patent/CN117630242A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111579706A (en) | Detection method of impurities in hydrolysis of brivaracetam | |
| CN113390983B (en) | Detection method for simultaneously determining 3 impurities in Voranolan fumarate | |
| CN112730641B (en) | Ion chromatography determination method of N-methylpiperazine | |
| CN117630242A (en) | Method for separating and detecting impurity a in clotrimazole intermediate by high performance liquid chromatography | |
| CN114354800B (en) | Method for analyzing acetyl bromide content in cefuroxime axetil | |
| CN114354801B (en) | Analysis method for contents of three aminopyridine isomers in (R) -3-Boc-aminopiperidine | |
| CN113219089B (en) | Method for detecting urea by post-column derivatization-liquid chromatography | |
| CN113109473B (en) | Formaldehyde derivatization test method | |
| CN114264765B (en) | Analytical method for determining related substances in glimepiride intermediate by utilizing HPLC | |
| CN115774061A (en) | Method for detecting acetic acid in 1-cyclohexyl piperazine | |
| CN113607833B (en) | Content analysis method of indoxacarb intermediate | |
| CN107976489B (en) | Method for determining residual pyridine in pregabalin | |
| CN117630202B (en) | Method for detecting buspirone hydrochloride intermediate impurities | |
| CN112305100B (en) | Method for detecting content of genotoxic impurity benzyl bromide in medicine | |
| CN115420826B (en) | Separation detection method for impurities in o-ethoxybenzoic acid as starting material in sildenafil citrate bulk drug | |
| CN113884584A (en) | Method for detecting content of flurbiprofen and/or flurbiprofen axetil | |
| CN118090987A (en) | Detection method for sulfonate impurities in michaux ammonium chloride | |
| CN117630247A (en) | Method for simultaneously determining multiple impurities in piperidine-4-formamide | |
| CN117665181A (en) | Method for detecting ammonia in methylamine alcohol | |
| CN117825547A (en) | Isolation assay of clotrimazole intermediate Z 1 High performance liquid chromatography of related substances in (B) | |
| CN117517528A (en) | Detection method of related substances in intermediate of ponatinib | |
| CN116413344A (en) | Quality control method and application of reagent | |
| CN117825546A (en) | GC-MS method for separating genotoxic impurities in clotrimazole and measuring content thereof | |
| CN118067860A (en) | Detection method of related substances in epalrestat tablet | |
| CN114113388A (en) | Method for detecting related substances in 4-oximinomethyl-1-naphthoic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |